The long-term goal of this investigation is to understand the molecular mechanisms underlying Mendelian inherited deficiency of mitochondrial multienzyme complexes. The model system for study is maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that produces severe neurological damage and mental retardation. The enzyme deficient in MSUD, the mitochondrial branched-chain alpha-keto acid dehydrogenase (BCKAD) complex, is a multisubunit enzyme complex encoded by six genetic loci. Little is known about how a mutation in a single subunit effects the macromolecular assembly and function of the BCKAD complex. This information is essential to improve treatment including somatic gene therapy for patients. The following aims are proposed to study MSUD at the cellular and molecular levels: A) Novel mutations in MSUD will be identified by the polymerase chain reaction (PCR), single strand conformation polymorphisms (SSCP) and nuclease protection methods. These studies will delineate genetic diversity, prevalence and origins of MSUD mutations. B) The functional consequences of MSUD mutations on the assembly and function of the BCKAD complex will be studied using efficient prokaryotic and eukaryotic expression systems. These studies will elucidate the biochemical mechanism responsible for thiamine- responsiveness in certain MSUD patients. C) The promoter function of the BCKAD complex genes will be studied. The cis-acting elements will be defined using the luciferase reporter assay. The transcription factors will be studied by DNase I footprinting and gel retardation assays. These approaches will facilitate detection of MSUD mutations in the promoter region, and test the hypothesis that the BCKAD complex genes are coordinately regulated in the cell. D) A stable retroviral-mediated gene transfer will be developed to correct the MSUD phenotype. A series of LN retroviral vectors will be used to transfer normal recombinant genes into MSUD lymphoblasts, human hepatoma and hematopoietic cell lines and rat primary hepatocyte cultures. The degree and duration of expression of the recombinant gene will be studied. The efficiency of eukaryotic and viral promoters in different cell types will be evaluated. These experiments may form the basis for development of somatic gene therapy for MSUD.